VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). Upon secretion into plasma VWF circulates in the form of various species with different molecular sizes. These VWF molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. VWF can be usually found in plasma as one dimer up to multimers consisting of 50-100 dimers (Ruggeri et al. Thromb. Haemost. 82: 576-584, 1999). The in vivo half-life of human VWF in the human circulation is approximately 12 to 20 hours.
The most frequent inherited bleeding disorder in humans is von Willebrand's disease (VWD), which can be treated by replacement therapy with VWF containing concentrates of plasmatic or recombinant origin. Due to the short half-life of VWF in blood there is a strong need to develop VWF concentrates with a prolonged in vivo half-life of VWF. The same applies to FVIII, which has also a relatively short in vivo half-life of approximately 8 to 12 hours requiring frequent re-dosing for patient treatment of bleeding disorders associated with functional defects of or deficiencies of at least one of FVIII and VWF.
In the prior art it has been described that recombinant VWF (rVWF) produced in an eucaryotic cell culture is more intact and less proteolytically degraded than plasma-derived VWF (Fischer et al., FEBS Lett. 375: 259-262, 1995). EP 0 784 632 describes a method for isolating highly pure VWF by purifying recombinant VWF using anion exchange chromatography. Methods for a large scale production of homogenous and structurally intact VWF are also known in the art (Schlokat et al., Biotechnol. Appl. Biochem. 24: 257-267, 1996; Fischer et al., CMLS 53: 943-950, 1997). Recombinant VWF has been characterized by using canine, murine, and porcine models of von Willebrand's disease (VWD) (Turecek et al., Blood 90: 3555-3567, 1997; Roussi et al., Blood Coag. Fibrinol. 9: 361-372, 1998; Schwarz et al., Haemophilia 4: 53-62, 1998; Schwarz et al., Semin. Thromb. Hemost. 28: 215-225, 2002). WO 00/49047 describes a method for producing a VWF preparation by treating pro-VWF with thrombin. A method for purifying proteins that bind to VWF by using a rVWF immobilized on a carrier is disclosed in WO 98/25969. The pharmaceutical use of plasma derived and recombinant VWF pro-peptides (pro-VWF) for treating blood coagulation disorders is described in EP 0 977 584. U.S. Pat. No. 6,037,452 describes the binding of FVIII and factor IX (FIX) to a poly(alkylene oxide) through a linker or a coupling agent. In EP 0 774 261 it is shown that the use of recombinant VWF having a prolonged biological in vivo half-life stabilizes FVIII in the blood of a mammal and induces the production of endogenous FVIII. Nevertheless, there exists a need for patients having VWF- or FVIII-based bleeding disorders to further increase the in vivo half-life of VWF and FVIII.
VWF is known to stabilize FVIII in vivo and, thus, plays a crucial role to regulate plasma levels of FVIII and as a consequence is a central factor to control primary and secondary hemostasis. It is also known that after application of therapeutic products containing VWF an increase in endogenous FVIII:C to 1 to 3 units per ml in 24 hours can be observed demonstrating the in vivo stabilizing effect of VWF on FVIII.
A strong need exists for a new substance for widening the treatment spectrum for deficiencies in coagulation FVIII also known as hemophilia A and/or qualitative or quantitative deficiencies of VWF also known as VWD. Due to a lack of functional VWF, patients with VWD have a secondary defect of FVIII represented by FVIII plasma levels below normal. Depending on the type of VWD and the severity of the diseases these FVIII levels can vary but are generally measurably lower than the FVIII plasma level found in healthy humans.
Thus, the present invention provides a novel system for prolonging the in vivo half-life of VWF and/or of FVIII in the blood of a mammal. It is a further object of the present invention to provide methods for the improved treatment of bleeding disorders associated with functional defects of or deficiencies of one or both of FVIII and VWF.